A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. This nutrient sensing hexosamine signaling pathway is conserved from nematodes to man. A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes, suggesting that perturbation of this pathway results in disease. We continue to explore the many functions of this signaling pathway in modulating aspects of nutrient sensing, development, and metabolic functions. In collaboration the Hanover lab (NIDDK), we showed that the C. elegans genome encodes the two evolutionarily conserved enzymes that mediate O-GlcNAc cycling, with the genes called ogt-1 and oga-1. We previously characterized knockout alleles of ogt-1 and oga-1 genes. Using a combination of genomic expression arrays and chromatin immunoprecipitation (ChIP) we are looking for genes that respond to nutrient flux differently in the mutants with the hope of identifying pathways of importance. The expression analysis has revealed widespread de-regulation of gene expression in the mutants, identify affected pathways including longevity and aging. We have tested these pathways in the mutants and find alteration in function that are consistent with the gene expression patterns we observe. From the ChIP studies, we have identified a discrete number of genes associated with O-GlcNAcylated proteins. These associations are pronounced at the promoters of the genes and show some overlap with ChIP signals using RNA PolII antibodies. We are currently investigating the functional roles, if any, of these restricted O-GlcNAc chromatin marks. These marks have the potential to link nutritional flux in the cell directly to gene regulation, offering a novel insight into the role of O-GlcNAc cycling in animal physiology and development. In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. During the past year, we have demonstrated that disruption of O-GlcNAc cycling perturbs nucleotide sugar pools and complex glycans in the organism. We have also begun to explore the utility of C. elegans in modeling rare human genetic disorders of metabolism. In collaboration with Dr. Semple (Univ of Cambridge, UK), we have initiated a proof-of-principle study using the DAF-2 insulin-like receptor in the worm to model mutations in the human insulin receptor (INSR). Combining bioinformatic analysis and in vivo assays, we are testing mutations located throughout DAF-2 for phenotypic consequences and correlating those changes with human disease alleles. We have exploited the relatively quick and easy forward genetics, genome editing, and phenotypic assays of the C. elegans system to gain insight into human insulin receptor functional domains, including creating an allelic series of mutations in a single amino acid residue that mimics human disease severity. In collaboration with Drs. Marta Kostrouchova and Kostrouch (Charles University, Prague) we have identified a protein in C. elegans that is related to human perlipins, proteins that associate with lipid droplets and regulate their metabolism. No prelipin homolog had previously been identified in C. elegans, raising the possibility that fat droplet regulation and metabolism in the nematode was different. Our results suggest an evolutionarily conserved, perlipin-like protein family is regulating lipid droplets in metazoan.